System and process for separating a material

ABSTRACT

Disclosed is a system to separate, enrich, and/or purify a cellular population from a biological tissue, such as a tissue sample. For example, an adipose tissue sample can be acquired and disrupted. The disrupted tissue sample can then be separated and purified. The separated components can include multipotent, pluripotent, or other cell populations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of both U.S. Provisional ApplicationNo. 61/032,619, filed on Feb. 29, 2008 and U.S. Provisional ApplicationNo. 61/078,178, filed on Jul. 3, 2008. The disclosures of the aboveapplications are incorporated herein by reference.

FIELD

The present disclosure relates generally to separation of a selectedcomponent from a multi-component biological material.

BACKGROUND

The statements in this section merely provide background informationrelated to the present disclosure and may not constitute prior art.

Various cellular (or biological) materials can be used to assist aperson in a healing or recovery process. Pluripotent cells, multi-potentcells, stem cells, or fully differentiated cells can be applied to apatient for providing therapy to the patient. For example, stem cellscan be applied to a patient to assist in healing an affected areathrough differentiation of the stem cells. In addition, stem cells canbe applied to an area of the patient that may be damaged due to injury,chemotherapy, or radiation therapy to assist in regeneration of theaffected cells.

Stem cells can be acquired from various sources. Selected sources caninclude autologous sources, where the patient is a donor or self donor.The autologous source of the stem cells can include various tissues,such as adipose tissue. Adipose tissue can be used as a source of cells,such as stem cells or vascular endothelial cells that can be separated,concentrated, or purified from the fat cells.

SUMMARY

A selected cell population from a selected tissue sample, such asadipose tissue, can be separated, enriched, and/or purified. Theselected cell population can include pluripotent, multipotent, or stemcells or other cells having therapeutic value for use in a therapy. Thepopulation can be collected via aspiration or excision of a tissuesample and further processed for separation and purification of theselected cells from the tissue sample. Therapeutic non-cellular material(for example extracellular matrix) can also be separated and purified.The tissue sample can include adipose tissue, mucosa, muscle, blood, orother appropriate tissue sources.

The tissue sample can be acquired from a patient and separated in anefficient and time effective manner for application to the same patient(i.e. autologous). Use of autologous cells can reduce possible rejectionand reduce contamination issues. Further, autologous cells can provide agenetic match to the patient.

A process for acquiring a tissue sample, according to variousembodiments, can generally include aspirating or excising adipose tissuefrom a patient. The aspirated or excised adipose tissue can then bedisrupted, macerated, and/or pulverized. The disrupted tissue can thenbe separated using a separation system as discussed herein. In theseparation system, the disrupted tissue can be suspended in a selectedbiologically acceptable solution that can aid in separation orenrichment of a selected fraction of cells.

A system for separating the selected fraction of cells from the tissuesample can include a container that is operable to rotate around acentral axis so that the tissue sample is moved towards or pressedagainst an outer wall (such as centrifugal forces) and separated basedupon size and/or density. Areas of different size or density can beformed between the central axis and the outer wall. The separationdevice can also have a separate concentric ring or collection area thatcan collect a selected fraction of the material, such as a selected cellfraction, and a closure valve for the collection area. Disruptiondevices, according to various embodiments, including those discussedherein, can be used to disrupt the tissue sample prior to the separationof the selected fraction in a separation system.

Although the present disclosure may refer to aspirate or lipoaspirate,it will be understood that any appropriate tissue sample may be used,unless specifically indicated otherwise. For example, muscle or mucosatissue can be excised, disrupted, and separated as discussed herein. Inaddition, adipose tissue may also be excised rather than only aspirated.

In addition, a selected fraction, such as a selected cell fraction, caninclude stem cells. It will be understood, however, that stem cells canbe used herein, unless otherwise specified, to indicate cells that areundifferentiated or incompletely differentiated and able todifferentiate into more than one cell type. For example, a stem cell maybe able to differentiate, depending upon various factors, into one ormore distinct cells. Certain populations of cells, however, may onlydifferentiate into one, two, or any discrete number of cell types. Also,a selected fraction can include cells that are unlimited or limited intheir differentiation possibilities. Undifferentiated cells can alsoinclude pluripotent or multipotent cells. These cells can differentiateinto an appropriate number of cell types.

Further areas of applicability will become apparent from the descriptionprovided herein. It should be understood that the description andspecific examples are intended for purposes of illustration only and arenot intended to limit the scope of the present disclosure.

DRAWINGS

The drawings described herein are for illustration purposes only and arenot intended to limit the scope of the present disclosure in any way.

FIG. 1 is a flowchart illustrating a general process for tissueseparation;

FIG. 2 is a perspective view of a tissue disrupter, according to variousembodiments;

FIG. 3A is a plan view of a tissue disrupter, according to variousembodiments;

FIG. 3B is a cross-sectional view of the disrupter of the FIG. 3A;

FIG. 4A is a frit assembly, according to various embodiments;

FIG. 4B is a plan and transparent view of the assembly of FIG. 4A;

FIG. 5 is a perspective view of a tissue disrupter, according to variousembodiments;

FIG. 6 is a plan view of a tissue disrupter, according to variousembodiments;

FIG. 7 is a tissue disrupter, according to various embodiments;

FIG. 8 is a plan view of the tissue disrupter of FIG. 7;

FIG. 9 is a plan transparent view of the tissue disrupter of FIG. 7;

FIG. 10 is a cross-sectional view of a tissue isolator, according tovarious embodiments;

FIG. 11 is a plan view of a tissue isolator, according to variousembodiments;

FIG. 12 is a plan perspective exploded view of a tissue isolator,according to various embodiments;

FIG. 13 is a cross-sectional view the tissue isolator of FIG. 12;

FIGS. 14A-14C are various detailed views of the tissue isolator of FIG.12;

FIG. 15 is a tissue isolator, according to various embodiments;

FIG. 16A-16D are various detailed views of the tissue isolator of FIG.15.

FIG. 17 is a cross-sectional view of a tissue isolator, according tovarious embodiments;

FIGS. 18A-18G illustrate a separation procedure of a tissue sample,according to various embodiments.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is notintended to limit the present disclosure, application, or uses.

With reference to FIG. 1, a flow chart 20 illustrates a general processof acquiring a selected fraction of cells from a tissue sample. Aselected fraction can include a cell population, such as stem cells orother undifferentiated cell populations, as discussed above. Accordingto various embodiments, the process starts at Start block 22. Anyappropriate tissue sample, (e.g. adipose tissue) can be acquired inblock 24. If the selected tissue is adipose tissue, it can be acquiredin various techniques, such as during liposuction, aspirating adiposetissue from a selected source, or other appropriate techniques. Forexample, during a prosthesis implantation, adipose tissue can be excisedor removed from the patient near or at the incision for the prosthesisimplantation. Therefore, aspiration of tissue is merely an exemplaryprocess for withdrawing or removing tissue from a patient.

The tissue sample can be mixed or suspended with an appropriate additivein optional block 25. Additives can include materials to adjust pH orfor chelating, such as citrate. A pH can be selected to be about 4 toabout 9, and can be selected to be about 4 to about 6. Other additivescan include lytic activity materials (for example, RBC lysis buffer),ion channel blockers, anti-apoptosis agents, protease inhibitors,density modifiers, chaotropic agents, osmolarity altering agents (todifferentially alter density and/or fragility of subpopulations ofcells), and/or detergents. These additives can alter pH, chelate variousions, etc. The additives, however, generally are enzyme free. Thus,enzymes are generally not, and need not be added to the tissue sample.In addition, any additive is optional and may not be added until thetissue sample is prepared for separation after processing. The solutionor the tissue can be maintained at about 35 to 45 degrees centigrade(C).

The tissue can be disrupted in block 26. The disruption of the tissuecan occur according to any appropriate technique or with any appropriatedevice, including those described further herein and may include addingadditives, as described above. Nevertheless, the disruption of thetissue can generally be performed to obtain a selected cluster orparticle size of the tissue. For example, it may be selected to obtainclusters of tissue (including cells and intercellular matrix) that areabout 0.1 mm to about 5 mm, in size including about 0.5 mm to about 2mm, and also including about 1 mm in average size. It will be understoodthat clusters of tissue may not be perfect spheres or other geometricshapes and therefore the average size can include a diameter dimension,a size defined by random measurement, a sieve measurement, or any otherappropriate measurement, such as microscopy, coulter counting, laserlight scattering and other techniques well known to those skilled in theart. In addition, an adipose tissue sample can be disrupted to about 0.5mm to about 2 mm, including about 1 mm in average cluster size.

A selected cell fraction or population can be prepared for separationfrom the tissue sample in block 28. The tissue can be suspended in aselected suspension material or otherwise prepared for separation. Forexample, a chelating agent, such as citrate, can be mixed with thedisrupted tissue. The citrate, or other chelating agent, can weaken thebonds between cells, cells and an extracellular matrix, and betweencells, the extracellular matrix, and other components in theextracellular matrix. Thus, citrate or other appropriate materials canassist in separation of a selected cell fraction. Further, citrate orother similar materials, such as chelating compounds, can assist orenhance separation without the addition of enzymes.

In addition, the solution can be maintained or provided at a selected pHfor purification or separation. It will be understood, the addition ofan acid or a base can be used to acquire a selected pH. Also, theaddition of various materials, such as the citrate, can alter or be usedto provide a selected pH of the material for separation.

The tissue can be suspended in a solution to assist with separation. Forexample, the tissue can be placed in an intermediate density solution(e.g. Ficoll® copolymers sold by GE HEALTHCARE BIO-SCIENCES AB ofSWEDEN) where adipose tissues can rise. The combination of theintermediate density solution and tissue can also be centrifuged, asdiscussed further herein. Centrifugation with an additional solution, inaddition to the tissue, is not necessary.

Other additives can also be added for various purposes as discussedabove. Materials, however, need not be added. This is true regardless ofwhether a material has already been added.

Once the tissue has been prepared for purification, the tissue can beplaced into a separation or purification device in block 30. Placing thetissue sample in the device in block 30 can include transferring thetissue cells and any additive materials previously added. It will beunderstood that additives need not be provided. The transfer of theprepared tissue to the separation device in block 30 can be performed inany appropriate manner. For example, the material can be transferred ina syringe (e.g. sterile syringe), a transfer container, a sterilesyringe, or any appropriate technique. Various transport systems caninclude those disclosed in U.S. patent application Ser. No. 11/222,303,filed Sep. 8, 2005, now U.S. Pat. No. 7,766,900, commonly assigned, andincorporated herein by reference.

The selected cell fraction can then be separated in block 32. Theseparation of the selected cell fraction can occur according to variousembodiments, including those discussed further herein. For example, theGPS™ separation system, sold by Biomet Inc. of Warsaw, Ind., USA can beused to separate the selected cell fraction from tissue. Exemplary buoyseparation systems are disclosed in U.S. patent application Ser. No.10/932,882, filed on Sep. 2, 2004, now U.S. patent application Ser. No.7,374,678, incorporated herein by reference. The buoy or separationdevice can be designed with an appropriate density or specific gravityfor stem cell separation from adipose tissue, or other appropriateseparations. Further, separation devices can include those discussedfurther herein.

The purified selected cell fraction can be transferred in block 34,according to various embodiments, automatically or manually to aselected or isolation area. For example, a separation device can be usedthat includes a separation area and a collection area where the purifiedselected cell fraction is collected substantially automatically during aseparation process. Therefore, the transfer of the purified cells inblock 34 is optional and other collection techniques can be used.Regardless, a collection of the selected cell fraction occurs in block36. The selected cell fraction that is purified can be collected in anyappropriate manner, as discussed further herein. In block 38, anoptional procedure can be performed with the selected cell fraction.Accordingly, the procedure may end at END block 40 after collecting thepurified selected cell fraction with or without performing a procedurewith the collected cells. Nevertheless, a selected procedure can beperformed, such as applying the selected cell fraction to a patient orusing the selected cell fraction in a selected therapeutic procedure. Itwill be understood, however, that the selected cell fraction can also beused for various other procedures, such as research, seeding a scaffold(i.e., building an ex-vivo structure), cell line creation, and the like.

With reference to FIG. 2, a disruption device can include a mechanicalpress 50. The mechanical disruption device 50 can be used to createdisrupted tissue 52 from aspirated or collected tissue 54. Themechanical disruption device 50 can include a collection area 56 intowhich the initially collected tissue 54, collected according to variousappropriate procedures, is placed. The collection area 56 can include afrit or screen 58 through which the undisrupted tissue can be forced tocreate the disrupted tissue 52. The undisrupted tissue 54 can be forcedthrough the frit 58 with a press portion 60 that is adapted orconfigured to fit within the collection 56 and force the undisruptedtissue 54 through the frit 58. A mechanical force can be applied to alever arm 62 to which the press portion 60 is connected to force theundisrupted tissue 54 through the frit 58. A fulcrum 64 can interconnectthe lever arm 62 of the press portion 60 with a second arm 66 which candefine or be connected with the collection area 56. After disruption,the disrupted tissue 52 can be processed according to the method 20,illustrated in FIG. 1, and further herein. It will be understood thatthe tissue can include an adipose tissue sample.

With reference to FIGS. 3A-4B, a dual syringe or reciprocating syringeand disruption frit assembly 70 is illustrated. The dual syringe systemcan include a first syringe assembly 72 including a cylinder 74 defininga wall and an interior and a piston rod and/or plunger 76. The pistonrod 76 with the syringe assembly 72 can terminate in a plunger orstopper member 78. To assist in reciprocation or movement of the plunger78, the syringe assembly 72 can include finger holds or holding members80 connected with the cylinder 74 and a second finger grasping portion82 interconnected with the rod 76.

A second syringe assembly 86 can be provided also including a cylinder88 and a rod portion 90 that terminates in a plunger 92. Similargrasping portions 94, 96 can be interconnected with a cylinder 88 androd 90. The two syringes can be interconnected with a frit member 100.The two syringe assemblies 72 and 86 can be used to move a sample 102through the frit assembly 100 between the two cylinder bodies 74, 88.The movement through the frit assembly 100 can disrupt the adiposetissue in a defined manner.

With reference to FIG. 4A, 4B, the frit assembly 100 can include twothreaded portions; a first threaded portion 104 and a second threadedportion 106. The two threaded portions 104, 106 of the frit assembly 100allow the first syringe assembly 72 and the second syringe assembly 86to engage a threaded coupling the frit assembly 100. It will beunderstood that other coupling mechanisms, such as a friction orinterference fit, can be provided to couple the syringes to the fritassembly 100. Thus, the first and second syringe assemblies 72, 86 canbe selectively connected with the frit assembly 100. This can also beused to allow for loading or unloading of the tissue sample. Forexample, one or both of the syringe assemblies 72, 86 can bedisconnected from the frit assembly 100 and the tissue sample can beloaded into one or both of the syringe assemblies. As discussed furtherherein, the syringe assemblies 72, 86 can then be used to reciprocatethe tissue sample through the frit member 108 to disrupt the tissuesample, as discussed further herein.

A frit member or body 108 can include one or more throughbores orpassages 110 that allow material to pass, such as tissue, as the twosyringe assemblies 72, 86 are used to reciprocate the sample through thefrit body 108. The sample is forced through the holes 110 with thesyringe assemblies 72, 86. The reciprocation mechanically disrupts thetissue and an appropriate number of reciprocations can occur to achievea selected disruption of the tissue sample. Furthermore, the holes 110in the frit body 108 can be provided in any appropriate dimension, suchas a diameter, to achieve a selected disruption of the fat. Therefore, adual or reciprocating syringe assembly 70 can also be used to disruptthe tissue sample in block 26 of the method 20. Again, the tissue samplecan include an adipose tissue sample from an appropriate source.

The holes 110 defined in the frit body 108 can include any appropriatedimension. For example, the frit holes can include a dimension of about0.02 inches to about 1 inch, including about 0.04 inches to about 0.5inches, such as about 0.25 inches. The dimension of the holes 110 can beany appropriate dimension and may vary depending upon the cell type tobe enriched or concentrated from the tissue sample. Also, the hole sizecan be provided according to the source of the tissue sample to bedisrupted. For example, a lipoaspirate originated sample may bedisrupted with a smaller opening to achieve a selected particle size asopposed to a sample from another source, such as a sample from anabdominoplasty.

Moreover, a kit including a plurality of the frit assemblies 100 caninclude multiple frit bodies with different holes of differentdimensions. Therefore, a user can select an appropriate frit dimensiondepending upon the source of the adipose tissue. Moreover, an adiposetissue that is being disrupted can be moved through a plurality of fritassemblies. The kit can include a plurality of the frit assemblies 100including hole dimensions that may be progressively smaller so that theadipose sample can be moved through progressively smaller holedimensions to progressively increase disruption of the fat or adiposetissue sample. For example, as discussed above, a first frit assembly100 including a first selected hole dimension can be interconnected withboth of the syringe assemblies 72, 86. The tissue sample can bedisrupted by reciprocating the syringe assemblies 72, 86 and after aselected period of time or cycles of reciprocation, the first fritassembly can be replaced with a second frit assembly having holes 110 ofa different dimension. The syringe assemblies 72, 86 can then bereciprocated to disrupt the tissue with the different hole dimensionsize for a selected period of time or number of cycles. This can berepeated until selected tissue sample particle size or disruption sizehas been achieved.

According to various embodiments, a single syringe, as opposed to a dualsyringe, with an appropriate frit assembly can also be used fordisruption. A single syringe can operate similarly to the duel syringe,save that the material can be expressed through the frit to a collectioncontainer or area. In addition, the frit assembly, according to variousembodiments, can include a single passage or orifice for disruption.Examples of a single orifice frit can include an aspiration needle.Various embodiments can include single plungers, as discussed herein,with various mechanical assist mechanisms. Various frits of differingpassage diameters can be provided for appropriate disruption of thesample.

According to various embodiments, a disruption assembly 150 isillustrated in FIGS. 5 and 6. The disruption assembly 150 can include asingle pass, non-reciprocating bone cement gun configuration. Varioussuch bone cement gun assemblies include the Optigun™ assembly sold byBiomet, Inc. of Warsaw, Ind., USA as a part of the Optivac™ CementMixing System. Briefly, the bone cement gun assembly 150 can include amain handle portion 152 and a power actuating handle 154. Generally, thepower handle 154 can move relative to the main handle 152 to cause amovement of an actuating rod 156. The connection of the handles 152, 154and the rod 156 can be any appropriate connection. For example, a pawland ratchet system can be used to advance the rod 156 in a selecteddirection, such as the direction of arrow A towards an exit end of 158of the assembly 140. A release mechanism can be depressed or moved toallow withdrawal or movement of the rod 156 in a direction of an arrow Btowards the handle assembly 152. Movement of the rod 156 towards thehandle assembly can be performed via manual movement or automaticmovement, such as pulling on a retraction handle 160.

The rod 156 can terminate in a plunger 162 held within a canister ortube 164. Near the exit end 158 of the tube 164 can be a frit ordisruption assembly 168. A sample of tissue 166, such as adipose tissue,can be introduced into the tube 164 for purposes of disruption. Thesample of tissue can be introduced into the tube 164 in various manners.For example, the frit assembly 168 can be removed or the entire tube 164can be removed from the trigger body. In this way, a sample can beintroduced into the tube 164 for disruption or extrusion through thefrit assembly 168.

The frit assembly 168 can include a frit body 170 that defines one ormore passages or holes 172. The passages 172 can include any appropriatedimension, such as those discussed above. As the assembly 150 isactuated, the plunger 162 can move the adipose tissue sample 166 throughthe holes 172 defined in the frit assembly 168. This can cause amechanical disruption of the sample 166 as it passes through the fritassembly 168.

As discussed above, the frit assembly 168 can include any appropriatefrit or hole dimensions. Also, the holes, according various embodiments,can be provided in any appropriate shape or configuration. Further, thefrit assembly 168 can be removeable, such as via a threadedinterconnection with threads 174 so that the frit assembly 168 can beapplied or removed from the tube 164. This can allow for introduction ofthe sample into the tube 164 or exchange of the frit assemblies 168. Forexample, a kit can include a plurality of the frit assemblies 168, eachincluding different dimensions of the throughbores 172. The tissuesample 166 can be pressed through a first frit assembly 168, the firstfrit assembly can then be removed, the sample replaced in the tube 164,a different frit assembly positioned on the tube 164, and the sampleagain pressed through the new frit assembly. Therefore, the tissuesample can be progressively and increasingly disrupted as it movesthrough a plurality of the frit assemblies.

With references to FIGS. 7-9, a disruption assembly 190 is illustrated.The disruption assembly 190 can include portions that are similar to thedisruption assembly 150 and similar reference numerals are used toidentify these portions and discussed again only briefly. For example,the disruption assembly 190 can include a tube 164 that has a terminalor exit end 158 and a frit assembly 168 positioned near the terminal orexit end 158. The frit assembly 168 can include a frit body 170 thatdefines one or more throughbores or holes 172. As discussed above, anaspiration needle could be considered one very long hole.

The disruption assembly 190 can include a rod 192 that defines anexternal thread 194. The external thread can engage an internal thread196 defined in an end cap or end portion 198 of the disruption assembly190. The end cap 198 can be removeable from the tube 164 via a threadedengagement 200. The rod 192, however, can be rotated in an appropriatedirection, such as the direction of arrow C to advance the rod towardsthe exit end 158 that, in turn, can push a plunger 202 towards the exitend 158. A sample 204, such as an adipose tissue sample, can be pressedthrough the holes 172 defined by the frit body 170 in the frit assembly168. The rod 192 can also be rotated in a counter direction, such asgenerally in the direction of arrow D, to move the plunger 202 towardsthe end cap 198. This can allow the plunger 202 to be moved back andforth within the tube 164 and can allow for filling of the tube 164,such as with removal of the frit assembly 168. Further, as discussed inrelation to the disruption assembly 150, a kit can include a pluralityof the frit assemblies 168 each including holes 172 of different sizes.Therefore, the sample 204 can be pushed through a frit assemblyincluding a first hole size and reloaded into the tube 164 and pushedthrough a second frit assembly including a second hole size.

Regardless of the configuration of the disruption assembly, it can beselected to disrupt a tissue sample to a selected particle or clustersize. For example, the cluster sizes can be selected to be anyappropriate dimension, such as those discussed above. Further, asdiscussed in relation to the various disruption assemblies, one or morepasses through one or more frit assemblies may be selected to achieve anappropriate particle size. Therefore, it will be understood that adisruption can include one or more passes through a frit assembly andvarious procedures, such as quality control procedures, can be used toensure that an appropriate particle size is achieved. Therefore, thedisruption assemblies, according to various embodiments, can be providedalone or in combination with other disruption assemblies to achieve anappropriate particle size, such as those discussed above.

Regardless of the disruption assembly provided, once disruption occursthe disrupted sample can be further separated or purified to acquire aselected component, such as a selected cell population. For example, apluripotent or stem cell population can be extracted from the tissuesample (e.g. disrupted adipose tissue). Various techniques can be usedto separate the pluripotent or stem cells from the disrupted tissueincluding those discussed further herein.

With reference to FIG. 10, a tube or separation assembly 220 can beused. A separation assembly 220 can include a tube 222 and a separationbuoy system 224. The buoy system 224 can include a first buoy member 226and a second buoy member or isolator portion 228. The first and secondbuoy portions 226, 228 can be fixed relative to one another or moveablerelative to one another. Further, either or both of the buoy assemblyportions 226, 228 can be tuned to a selected density or specific gravityto allow for a collection area 230, defined between the two buoyportions 226, 228, to be provided at an equilibrium position that caninclude the selected cell population, such as the pluripotent cells froman adipose tissue sample. A syringe 240, or other appropriate collectionor extraction device can be used to collect the material from within thetube 222. Appropriate separation tubes can include the GPS II™separation system sold by Biomet, Inc. of Warsaw, Ind., USA.

With reference to FIG. 11, according to various embodiments, aseparation tube assembly 280 is illustrated. The separation tubeassembly 280 can include a separation tube 282 that includes aseparation buoy assembly 284. The buoy assembly 284 can include a mainbuoy portion or buoy 286 and a second buoy portion or isolator 288. Theisolator 288 can be separated from the buoy portion 286 by a distance todefine a collection area 290. Within the collection area 290, two ormore collected materials or material areas 290 a, 290 b can be formed.

Again, the assembly of the buoy portion 286 and the isolator 288, orselected portions thereof, can include a tuned density or specificgravity to position the collection area 290 at a selected equilibriumposition of a sample that is positioned within the tube 282. Also, anappropriate collection or extraction syringe or device 294 can be usedto collect material from the tube 282. Selected separation tubes caninclude the GPS III ™ system sold by Biomet, Inc. of Warsaw, Ind., USA.Further exemplary buoy separation systems are disclosed in U.S. patentapplication Ser. No. 12/101,594, filed on Apr. 11, 2008, now U.S. Pat.No. 7,992,725, incorporated herein by reference.

It will be understood, however, that appropriate separation tubes can bedesigned in appropriate manners to allow for separation of a selectedpopulation from the tissue sample. Further, the separation tubes, suchas the separation tubes 220, 280 can be provided with separation orpower systems, such as a centrifuge system. The centrifuge system canprovide a separation force or gravitational force to the sample and thebuoy positioned within the tubes to allow for separation of the sampleposition within the tubes.

With reference to FIGS. 12 and 13, a separation system 300 isillustrated. The separation system 300 can include three maincomponents. The three main components can include an outer chamber orhousing 302, an inner chamber or container 304, and a drive base 306.The drive base 306 can include controls 308 and an internal motor orpower source to drive a rod or axle 310. The outer housing 302 cansurround all or part of the drive base 308 and the axle 310. The motorbase 306 can spin the inner container 304 around a central axis 312defined by the axle 310 and a portion of the inner chamber 304. Asdiscussed further herein, rotation around the axis 312 can cause a forcewithin the chamber 304 to create a separation of material positionedwithin the chamber 304.

The outer housing 302 can engage or include a lid 314 that includes aconnection or port 316. The port 316 can interconnect with a conduit orcannula 318 to allow for introduction or extraction of a material from amain container area 320 of the inner chamber 304. The main containerarea 320 can be used to hold a material before and during a portion ofthe separation in an isolation process, as discussed further herein.Further, the lid 314 may include additional ports for access to otherareas of the housing 302 or the container 304, such as an isolationarea, including an annular isolation ring 322.

The inner container 304 can include the main container area 320 and theisolation area 322 separated by a wall portion 324, a sealing member,such as an o-ring 326, and a lid 328. The lid 328 can be held relativeto the wall portion 324 and the sealing member 326 in any appropriatemanner, such as by gravity or a biasing mechanism. The lid 328 can beallowed to move relative to the central cannula 318 according to anyappropriate method, such as via a bearing 330. The lid 328 can moverelative to the o-ring 326 at a selected time, as discussed furtherherein, to allow for an opening between the main chamber area 320 andthe isolation area 322. The movement of the lid 328 can be along theaxis 312 or can include a movement of a portion of the lid 328 to allowfor an opening between the main chamber 320 and the isolation area 322.

An optional vent 325, 325′ for the isolation area 322 on the side of abarrier wall 344 (FIG. 14A) opposite an extraction port 346 (FIG. 14A)can be provided, according to various embodiments. The vent 325 canoptionally open to the space above the flexible lid or liner.Alternatively, the vent 325′ can optionally open to the central chamber320. The vent 325, 325′ can allow air to enter the isolation area 322 toreplace the volume of material withdrawn and for air to alternatelyescape if the material is jetted in and out to improve cellre-suspension and recovery during material collection, such as cellularmaterial. The vent 325, 325′ can also be used as a port for introductionof a stream of wash solution. The vent 325, 325′ can also include atwo-way or one-way valve that is manually or automatically operatedbased upon pressure differentials or other mechanism.

The main chamber area 320 can include any appropriate volume ordimension. For example, a wall 332 of the main chamber area can definean angle 334 between the central axis 312 and the wall 332. The anglecan be any appropriate angle, such as between about zero degrees andabout ninety degrees, but generally less than ninety degrees. The angle334 can also include about one degree to about forty degrees, andfurther include about ten degrees to about thirty degrees, and may alsoinclude about twenty degrees. The angle 334 can be provided or selectedto assist in a separation or movement of material from the main chamberarea 320 to the isolation area 322.

In addition, the inner chamber 304 can be formed of any appropriatematerials, such as a material that includes a selected co-efficient offriction relative to the material positioned within the inner chamber304 for separation. For example, an adipose tissue sample can bepositioned within the inner chamber 304, such as within the inner area320, for separation. It can be selected to include a co-efficient offriction between the adipose sample or a portion of the adipose sampleto achieve a selected separation at a selected period of time and/orunder a selected force, such as a centrifugal force.

With further reference to FIGS. 14A-C, the inner chamber 304 is furtherillustrated. The inner chamber 304 can include the isolation area 322separated from the main containment area 320. The isolation area 322 caninclude a complete or a partial annular ring surrounding the innerchamber or area 320. In addition, multiple isolation areas 322 can beprovided, for example as multiple concentric annular rings (notspecifically illustrated). The multiple isolation areas 322 could beused to isolate multiple fractions of intermediate or multiple densities(e.g., buffy coat from whole blood). A sealing portion or valve can beprovided between each or any appropriate group of the multiple isolationareas. For example, the lid 328 or liner can include multiple regions toact as sealing portions between each of the concentric isolation areas.In this way, multiple specific and discrete materials can be separatedand/or concentrated.

When a partial annular ring is provided, the isolation area 322 canextend from a first end 340 to a second end 342 that can be less than360 degrees around the central axis 312 from the first end 340. Asdiscussed further herein, the inclusion of the first end 340 and thesecond end 342 separated by a wall or barrier portion 344 can allow forsubstantially complete extraction of a material from the isolation area322. Briefly, if the wall 344 were not present it may be difficult toextract material from an area not adjacent to an extraction port, suchas the extraction port 346. The extraction port 346 can be positionednear or within a well or sump 348 that can further assist in removingmaterial from the isolation area 322. The inclusion of the wall 344allows for a vacuum to be created within the isolation area 322 relativeto the extraction port 346 and allow for further extraction orefficiency of extraction from the isolation area 322.

The extraction port 346 can be accessed from any appropriate location.Examples include, through the lid 328 of the inner container 304, thelid of the exterior chamber 314, or any other area through the housing302 or relative to the housing 302. Further, the extraction port 346 caninclude a cannula or connection portion 350 that extends from the port346 and can be interconnected with any appropriate portion, such as atube, extraction syringe, or any other appropriate mechanism. Materialfrom the isolation area 322 can be extracted for various purposes,including those discussed further herein.

With reference to FIGS. 15 and 16 A-D, a separation assembly 400 isillustrated. The separation assembly 400 can be substantially identicalto the separation assembly 300 except for various specifics of an innerchamber 402. Therefore, the outer housing 302 and the drive base 306 aresimply illustrated and will not be discussed in detail here. The innerchamber 402 can also be substantially similar to the inner chamber 304.For example, the inner chamber 402 can include the central axis 312, theisolation area 322, the optional vents 325, 325′, and the main innerchamber 320. Further, the various portions such as the wall 332 can beformed of similar materials and positioned at respective locations andorientations relative to other portions of the chamber 402.

The inner chamber 402, however, can differ from the inner chamber 304,illustrated in FIG. 13, at least by a lid 404 positioned relative to awall portion 406 of the inner chamber 402. The lid portion 404 can beformed of a substantially flexible material, or a least an external orperiphery portion 408 can be formed of a flexible portion. Further, theinner chamber 402 can be provided to not include the sealing member 326provided with the inner chamber 304. The flexible portion 406 of the lid404 can flex relative to the isolation area 322 at a selected time, asdiscussed further herein. Further, the flexible portion 408 of the lid404 can also form a seal relative to the portion 324 of a wall 406.Therefore, rather than having the lid 328 that can move relative to thesealing member 326, the lid 404 can define a flexible portion 408 thatcan act as a sealing portion relative to the upper wall portion 406 andalso flex relative to the upper wall 406. When the flexible portion 408flexes it can provide an opening over the wall 406, thus it can act as avalve.

The lid 404 can be designed to flex at a specific location or at aspecific region. It will be understood, however, that this is notnecessary. The lid 404 can be provided to generally flex an appropriateamount. For example, the lid may flex at a periphery or near a center ofthe lid. The lid 404 can be formed of appropriate materials and ofappropriate dimensions to allow the appropriate amount of flexing.

The lid 404 may also be provided as a liner over which an additional lidis provided. The additional lid or outer cover lid can provide a stop orlimit to the amount of flexing or movement of the lid liner 404, underthe outer lid. Accordingly, it will be understood that the lid 404 orany other appropriate flexible member and can be provided to allow anescape of a selected cell fraction into the isolation area.

The inner chamber portion 402 can also define the withdrawal port 346,the sump 348, and the withdrawal cannula 350. The isolation area 322 canfurther include the two ends 340, 342 separated by the barrier area 344.Accordingly, the inner chamber 402 can operate substantially similar tothe inner chamber 304, as discussed further herein, but can include theflexible portion 408 of the lid 404 rather than a separate and distinctsealing member, such as the sealing member 326.

With reference to FIG. 17, a separation assembly 450 is illustrated. Theseparation assembly 450 can include an outer housing 452 that cangenerally include a standard centrifuge system, such as the tissuecentrifuge system sold by Drucker or the CentraCL3 sold by ThermoScientific. The centrifuge system can include a selected drive system ormotor that can drive an axle or rod 454. The drive portion of theseparation system 450, however, can be substantially or relatively morepowerful than the drive system 306. Further, the outer housing 452 canbe more robust or heavier and allow for reduced vibrations duringseparation and rotation of the separation inner chamber 304, or anyappropriate inner chamber. The inner chamber 304 can be substantiallysimilar to the inner chamber 402 discussed above except that it can berotated by any appropriate motor or drive system. Therefore, theseparation chamber 304 can be driven at any appropriate separation ordrive speeds or for any appropriate length of time to allow forseparation of a selected material.

With reference to FIGS. 18A-18G, the separation system 300 can be usedto separate a selected component from a sample, such as in block 32 ofFIG. 1. It will be understood that the separation system 300, however,can include various portions, either alone or in combination, such asthe sealing member 326 or the flexible portion 408. Therefore, althoughthe separation system 300 is illustrated in FIGS. 18A-18G, anyappropriate separation system can be used and this is merely exemplary.Also, although the following discussion exemplary discusses separationof adipose tissue, any appropriate tissue can be separated. In addition,any appropriate cell fraction can be separated from the tissue sample.

As discussed above, a sample, such as an adipose tissue sample, can beagitated or disrupted according to various embodiments, including thosediscussed above. Once the tissue sample is appropriately disrupted, itcan be mixed with various materials. Materials that can be mixed withthe sample include citrate, a biologically acceptable acid, EDTA, orother appropriate materials. It will be further understood that amaterial or solution can be added at any appropriate time, such as priorto disruption of the sample, for pH adjustment, chelating, lysing, etc.It can be selected to create a solution 480 that can be placed withinthe main container area 320 of the inner chamber 304. The solution 480can include the sample, such as the adipose sample, and any appropriatematerials, such as citrate. The solution 480 can be adjusted to anyappropriate pH and can include chelating agents, such as citrate orEDTA. Regardless it can be selected to include with the tissue samplevarious extra cellular materials, such as extra cellular matrix.

Various materials, however, need not be provided. For example, noextrinsic enzymes or extrinsic collagenase need be added to the solution480. The disruption and the separation system can provide an appropriateseparation of the tissue sample. This can also provide a final productthat lacks extrinsic enzymes which can eliminate the need to attempt toremove selected enzymes. The absence of extrinsic enzymes can alsoeliminate concern of contamination or patient rejection. Althoughnaturally occurring enzymes may be present in the tissue sample, whichare not specifically removed, extrinsic enzymes are those added to thetissue sample whether artificially formed or not.

The sample can be placed into the inner chamber 304 in any appropriatemanner, such as through the entry port 316 from a supply vessel 482. Thesupply vessel 482 can include the syringes, as discussed above, or anyappropriate supply vessel. Further, as discussed above, the tissuesample can be provided from a patient to whom the selected material willbe supplied. In various embodiments, the selected cell population can besubstantially autologous. Once the solution 480 is positioned within theinner chamber 304, the drive base 306 can be activated to drive theinner chamber 304 in a selected manner, such as in a rotation around thecentral axis 312.

The rotation around the central axis 312 can initially cause thematerial to rise to a selected band within the inner chamber 304, asillustrated in FIG. 18C. The solution 480 can be moved to an appropriatelocation within the inner chamber 304 based upon the forces suppliedwith the rotation of the inner chamber 304 around the central axis 312.At a selected speed, a fat portion or oil portion 484 of the initialsolution or sample can float near the central axis 312 on top of orcloser to the central axis than an aqueous portion 486 of the initialsample, as illustrated in FIG. 18C. The initial separation of the fatportion 484 from the aqueous portion 486 can be achieved at a selectedrotational speed or separation force. For example, the initial rotationcan generate a force of about 10 to about 1000 times that of standardgravity on Earth (Gs). The rotation or centrifugation times can be anyappropriate time. According to various embodiments, the rotation timecan be about 60 seconds. A selected separation can occur, however,intraoperatively to allow for efficient autologous use of the selectedcell fraction.

For example, a separation of the material positioned within the innerchamber 304 can be achieved through one or more spinning speeds orseparation forces. Therefore, at an initial speed the fat portion 484can be separated from the aqueous portion 486. The aqueous portion 486can include various biological portions, such as cellular material,cellular matrix, and other appropriate materials. It will be furtherunderstood that the aqueous portion 486 can also include othermaterials, such as portions of whole blood, particularly if whole bloodis introduced with the tissue sample in the initial solution 480.

At a selected time or after a selected period of time at a firstseparation force, a cellular material and/or cellular matrix (i.e.selected cell fraction) 488 can migrate to a top portion or near theseal portion 324 of the inner chamber 304. A second rotation speed orforce can be about 300 Gs to about 3000 Gs. The migration of thecellular material 488 can be achieved after a selected period ofrotation or at a selected rotational force. For example, a higherrotational speed or higher force can be applied to achieve a migrationof the cellular material 488 towards the upper wall or the lid of theseparation container 304 near the isolation annulus 322.

The separation of the selected cell fraction 488 from the remainder ofthe solution, including the aqueous portion 486 and the fat portion 484,can be achieved with a change in speed of rotation, a change in force, atime change in spinning of the material, or any appropriate separation.Nevertheless, once the cellular material, which can include pluripotent,multipotent, or stem cells, cellular matrix, growth factors, and othermaterial, an isolation of the material can be achieved.

With reference to FIG. 18E, the lid 328 can move relative to the sealingmember 326 to allow an opening 490 so that the cellular material 488 canbe moved into the annular separation area 322. The lid 328 can moverelative to the sealing member 326 for any appropriate reason. Forexample, electrical and/or mechanical systems can be used to open ormove the lid relative to the sealing member 326. In addition, a selectedforce can be applied by the material within the separation chamber 320to urge or force the lid to move or flex a selected amount opening apassage and allowing the cellular material 488 to move into theisolation area 322. For example, a third speed or force can be appliedto the material within the separation chamber 304 such that it is forcedagainst the lid 328 to move the lid relative to the sealing member 326so that the cellular material 488 can move into the separation area 322.Therefore, the separation system 300 can allow for a substantiallyautomatic isolation of the material based upon forces, such ascentrifugal, created within the inner chamber 304, such as forcesapplied by the material positioned within the inner chamber 304 ascreated by spinning of the inner chamber 304 around the central axis312.

If a speed is selected to achieve an opening or movement of the lid 328relative to the sealing member 326, after a selected period of time, thespeed of rotation can be changed to allow for the lid 328 to move backand engage the sealing member 326 to seal the lid onto the sealingmember 326. The sealing of the lid 328 onto the sealing member 326 cansubstantially isolate the isolation area 322 from an area exterior tothe isolation area 322, including the main separation area 320.Therefore, the isolation area 322 can be used to collect a selectedmaterial, such as cellular material, based upon a density or specificgravity of the cellular material 488 and a gravitational separationrelative to the other material in the original solution 480.

With reference to FIG. 18G, once the separation has completed, thespinning of the inner chamber 304 can be ceased for extraction ofmaterial from the inner chamber 304. The components can remain separatedand may include the oil portion 484, the aqueous portion or purified fat486, and excess or extraneous material 498. The cellular material 488,within the isolation chamber 322, can then be extracted via anyappropriate extraction mechanism. For example, the selected cellfraction can be withdrawn via the extraction port or an extractionmember that is positioned into the isolation area 322.

The isolation area 322 can include a barrier wall 344 to interrupt acomplete annular ring of the isolation area 322. The barrier wall allowsonly a single path of extraction of material from the isolation area322. The extraction port can be positioned near or at the wall 344 toallow for an efficient extraction of substantially all of the materialpositioned within the isolation area 322. Accordingly, a vacuum drawn atthe wall 344 will urge substantially all of the material in theisolation area 322 out of the isolation area 322. It will be understood,as discussed above, that the optional vent 325, 325′ can be provided toassist or allow extraction of the material from the isolation area 322.

The extraction or withdrawal of the selected cell fraction from theisolation area 322 can therefore, include various processes such asre-suspending the cellular material within a liquid material that iseither positioned within the isolation area 322 from an external sourceor formed as a part of the separation. The re-suspension can beperformed through reciprocation of an extraction syringe 500 that canform a jet of material within the isolation area 322. The formation of ajet can cause a re-suspension of the selected cell fraction 488 withinthe isolation area 322.

The various processes can achieve an enriched extraction of the selectedcell population including stem cells, multipotent cells, or pluripotentcells from a tissue sample, such as adipose tissue. For example, thevarious processes, including the initial disruption to achieve aselected particle size, can allow for an efficient extraction ofselected material from the tissue sample. The extraction can beefficient even without the use of extrinsic enzymes. Further, theisolation area 322 positioned relative to a main area 320 can allow foran efficient and rapid isolation of the cellular material 488 from theother material positioned within the interior chamber 304. In addition,the isolation area 322, substantially separated from other areas andforming a substantially non-continuous annular ring, can allow forre-suspension and efficient extraction of the cellular material.Therefore, the separation of the material can be both efficient andproduce an extract that is highly enriched. The cell populationextracted can be extracted in an appropriate and high yield from thetissue sample for various purposes. The application of the extractedcells can then be made to the patient from whom the tissue sample wasacquired or to any other appropriate patient. The extracted cells can beprovided for various purposes, such as healing, tissue regeneration,cell replacement therapy, scaffold seeding, cell line establishment orother uses.

According to various embodiments, the separation system and derived orseparated selected cell fraction can be used to form an adipose-derivedtissue implant containing pluripotent cells in a collagen extracellularmatrix, comprising an extracellular matrix containing mechanicallydisrupted collagen substantially free of adipocytes and lipids andsubstantially or entirely free of extrinsic enzymes and comprised ofgreater than about 5% pluripotent cells. In addition, an adipose-derivedtissue implant, as discussed above, can be derived where the tissueparticle size is less than about 1 mm diameter. The implant may compriseblood components carried in the extracellular matrix, including growthfactors and plasma, white blood cells, red blood cells, and platelets;as well as pluripotent cells carried in the extracellular matrixincluding fibroblasts, endothelial cells, smooth muscle cells, mastcells, pericytes, fibroblasts, lineage committed progenitor cells, andpre-adipocytes.

The pluripotent cells can be carried in the extracellular matrix, andthe implant can be entirely or substantially free of extrinsic enzymeand the implant can be substantially free of adipocytes, and theisolated tissue of the pluripotent cell-containing extracellular matrixis comprised of single cells and clusters of up to about 0.10 cm indiameter. In addition, extracellular matrix may contain fragments ofconnective tissue, both free and associated with cells and cell clustersof diameters up to about 0.10 cm.

The adipose derived tissue implant containing pluripotent cells in acollagen extracellular matrix can be therapeutic for the variousclinical indications, such as: fat transplantation for soft-tissuecosmesis, cartilage repair for either acute cartilage lesions or forosteoarthritic joints, skeletal muscle repair, cardiac muscle repairfollowing a myocardial infarction, revascularization of critical limbischemia, fracture repair, spine fusion, avascular necrosis, bone voidfillers, augmentation of total joint arthroplasty, tendon reconstructionor repair, ligament reconstruction or repair, meniscus reconstruction orrepair, wound healing or combinations thereof.

The foregoing description of the embodiments has been provided forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention. Individual elements or features ofa particular embodiment are generally not limited to that particularembodiment, but, where applicable, are interchangeable and can be usedin a selected embodiment, even if not specifically shown or described.The same may also be varied in many ways. Such variations are not to beregarded as a departure from the invention, and all such modificationsare intended to be included within the scope of the invention.

1. A method of separating a selected cell fraction from a sample volume,comprising: disrupting the sample volume to obtain a sample particle ina selected particle size range different than a sample volume particlesize range prior to the disruption of the sample volume; separating theselected cell fraction from the sample particle, including, disposingthe sample particle in a first section of a separation container;applying a first centrifugal force to the sample particle in the firstsection of the separation container; collecting the selected cellfraction in a second section of the separation container; removing theselected cell fraction from the second section of the separationcontainer.
 2. The method of claim 1, further comprising: spinning theseparation container around a central axis to apply the centrifugalforce to the sample particle; moving the sample particle along aninterior wall of the first section of the separation container whilespinning the separation container; and transferring the selected cellfraction into the second section of the separation container whilespinning the separation container.
 3. The method of claim 1, furthercomprising: mixing the at least one of the sample volume or the sampleparticle with citrate, a biologically acceptable acid, or EDTA prior toseparating the selected cell fraction.
 4. The method of claim 1, whereindisrupting the sample volume to obtain a sample particle in a selectedparticle size range includes forming the sample particle to have anaverage particle size in a range of about 0.1 mm to about 5 mm.
 5. Themethod of claim 1, wherein disrupting the sample volume to obtain asample particle in a selected particle size range includes forming thesample particle to have an average particle size in a range of about 0.5mm to about 2 mm.
 6. The method of claim 1, wherein disrupting thesample volume to obtain a sample particle in a selected particle sizerange includes forming the sample particle to have an average particlesize of about 1 mm.
 7. The method of claim 1 further comprising:disrupting the sample volume substantially only with a mechanical forceapplied to the sample volume to obtain the sample particle.
 8. Themethod of claim 7, further comprising: obtaining the sample volume froma source body; and applying the selected cell fraction to the sourcebody.
 9. The method of claim 7, further comprising: obtaining the samplevolume from a volume of adipose tissue of a patient; wherein disruptingthe sample volume includes forming a plurality of sample particles. 10.The method of claim 1, further comprising: prior to applying acentrifugal force, forming a seal between the first section of theseparation container and the second section; and removing the seal frombetween the first section of the separation container and the secondsection of the separation container upon application of an opening forcedue at least in part to the applied centrifugal force.
 11. The method ofclaim 10, wherein applying the centrifugal force includes applying atleast a first centrifugal force and a second opening centrifugal force,wherein the second opening centrifugal force includes the opening force;wherein applying the second opening centrifugal force causes at least aportion of the selected cell fraction to exert a force on a lid thatcloses a passage between the first section to the second section to openthe lid to allow the selected cell fraction to move from the firstsection to the second section allowing the collecting of the selectedcell fraction.
 12. The method of claim 10, further comprising: flexing aportion of a lid to remove the seal from between the first section andthe second section with the second opening centrifugal force.
 13. Themethod of claim 10, further comprising: moving a lid from a first lidposition that is sealing the first section from the second section to asecond lid position that is unsealing the first section from the secondsection.
 14. The method of claim 1, further comprising: collecting theselected cell fraction in a second section of the separation containerincludes moving the selected cell fraction to a sump; and removing theselected cell fraction from the second section of the separationcontainer includes applying a suction to the sump to remove the selectedcell fraction.
 15. A method of separating a selected cell fraction froma sample volume, comprising: disrupting the sample volume to obtain aseparable sample having a sample particle in a selected particle sizerange; separating the selected cell fraction from the sample particle,including, disposing the separable sample having the sample particle ina first section of a separation container having a first wall extendingfrom a first bottom wall to an upper rim having an interior surface andan exterior surface; applying a centrifugal force to the sample particlein the first section of the separation container to cause both (1)collecting the selected cell fraction in a collection section positionednear the upper rim and at least contacting the exterior surface of thefirst wall, the collection section having a second wall extending from asecond bottom wall to the upper rim, and wherein the second bottom wallis nearer the upper rim than the first bottom wall and (2) moving asealing section that seals a collection volume within the collectionsection when in a closed position to an open position by flexing aflexible section to allow the sealing section to move to the openposition from the closed position; and removing the selected cellfraction from the collection section of the separation container. 16.The method of claim 15, further comprising: operating a drive motor tospin the separation container and the collection section simultaneouslyto apply the centrifugal force.
 17. The method of claim 16, furthercomprising: closing a housing to enclose all of the separationcontainer, the collection section, the lid, the sealing section, theflexible section, and the drive motor.
 18. The method of claim 15,further comprising: placing a lid assembly extending over both theseparation container and the collection section; wherein in the closedposition the sealing section selectively seals the collection volumefrom a container volume of the separation container; wherein at aselected spin rate the applied centrifugal force the sealing sectionmoves to the open position from the closed position; applying acentrifugal force to the sample particle in the first section of theseparation container to cause both (1) collecting the selected cellfraction in a collection section positioned near the upper rim and atleast contacting the exterior surface of the first wall, the collectionsection having a second wall extending from a second bottom wall to theupper rim, and wherein the second bottom wall is nearer the upper rimthan the first bottom wall and (2) moving a sealing section.
 19. Themethod of claim 15, wherein disrupting the sample volume to obtain aseparable sample having the sample particle in the selected particlesize range includes forming the sample particle to have an averageparticle size in a range of about 0.1 mm to about 5 mm.
 20. The methodof claim 15, wherein disrupting the sample volume to obtain a separablesample having the sample particle in the selected particle size rangeincludes forming the sample particle to have an average particle size ina range of about 0.5 mm to about 2 mm.
 21. The method of claim 15,wherein disrupting the sample volume to obtain a separable sample havingthe sample particle in the selected particle size range includes movingthe sample volume though a perforated member defining at least a throughbore.
 22. The method of claim 21, wherein moving the sample volumethough a perforated member defining at least a through bore includes:placing the sample volume in a sample containing volume having an endcovered by the perforated member; and moving a forcing member to forcethe sample volume through the perforated member to create the sampleparticle.
 23. The method of claim 15, wherein removing the selected cellfraction from the collection section of the separation containerincludes: drawing a vacuum within the collection section wherein abarrier wall defines a first end of the collection section on a firstside of the barrier wall and a second end of the collection section on asecond side of the barrier wall such that the barrier wall disrupts acomplete annular well of the collection section; wherein a vacuum isoperable to be formed near the first end of the collection section todraw substantially all material out of the collection section through aport.
 24. The method of claim 15, further comprising: connecting awithdrawal device to a passage defined through a housing that enclosesthe separation container and the collection section.
 25. A method ofseparating a selected cell fraction from a sample volume, comprising:forming a separable volume having a sample particle in a selectedparticle size range from the sample volume; disposing the formed sampleparticle in a first section of a separation container; applying a firstcentrifugal force to the sample particle in the first section of theseparation container while the first section is sealed relative to asecond section of the separation container to cause at least a portionof the selected cell fraction to move within the first section of theseparation container; applying a second centrifugal force to the sampleparticle in the first section of the separation container to cause asealing member to move from a sealed position to an unsealed position;applying a third centrifugal force to the sample particle in the firstsection of the separation container after causing the sealing member tomove to the unsealed position to allow at least the portion of theselected cell fraction to move into the second section of the separationcontainer; and removing at least a portion of at least the portion ofthe selected cell fraction from the second section of the separationcontainer.
 26. The method of claim 25, wherein applying the centrifugalforce includes spinning the separation container around a central axiswhich causes at least the selected cell fraction to move along aninterior wall of the first section of the separation container andtransfer at least the portion of the selected cell fraction into thesecond section of the separation container.
 27. The method of claim 25,further comprising: prior to applying the first centrifugal force,forming a seal between the first section of the separation container andthe second section of the separation container with the sealing member.28. The method of claim 27, wherein applying the first centrifugal forceand applying the second centrifugal force are the same force at a firsttime and a second time, respectively.
 29. The method of claim 28,wherein applying the second centrifugal force causes the seal member tomove to the unsealed position.
 30. The method of claim 29, whereinapplying the second centrifugal force includes causing at least aportion of the selected cell fraction to exert a force on the sealingmember to allow the selected cell fraction to move from the firstsection to the second section.
 31. The method of claim 25, whereinapplying at least the second centrifugal force includes flexing aportion of the sealing member to remove the seal from between the firstsection and the second section.
 32. The method of claim 25, furthercomprising: moving the selected cell fraction to a sump; and applying asuction to the sump to remove the selected cell fraction.
 33. The methodof claim 25, further comprising: moving the selected cell fraction to asump by ceasing the application of the first centrifugal force, ceasingthe application of the second centrifugal force, and ceasing theapplication of the third centrifugal force.
 34. The method of claim 25,further comprising: mixing the at least one of the sample volume or thesample particle with citrate, a biologically acceptable acid, or EDTAprior to at least one of forming a separable volume, disposing theformed sample particle in a first section of a separation container,applying a first centrifugal force, applying a second centrifugal force,or applying a third centrifugal force.
 35. The method of claim 25,wherein forming the separable volume having the sample particle in theselected particle size range includes forming the sample particle tohave an average particle size in a range of about 0.5 mm to about 2 mm.36. The method of claim 25, wherein forming the separable volume havingthe sample particle in the selected particle size range includesdisrupting the sample volume with only a mechanical force applied to thesample volume to obtain the sample particle.
 37. The method of claim 36,wherein disrupting the sample volume with only a mechanical forceincludes moving the sample volume though a perforated member defining atleast a through bore.
 38. The method of claim 37, wherein moving thesample volume though a perforated member defining at least a throughbore includes: placing the sample volume in a sample containing volumehaving an end covered by the perforated member; and moving a forcingmember to force the sample volume through the perforated member tocreate the sample particle.
 39. The method of claim 36, furthercomprising: obtaining the sample volume from a source body; and applyingthe selected cell fraction to the source body.